PROJECT SUMMARY Cells are exposed to many distinct stresses during their lives including extreme temperatures, altered osmolarity, hypoxia, free radicals, infections, or genotoxic insults. A harmful consequence of stress exposure is that the structures of essential cellular biomolecules can be damaged, and the damage can become deleterious if it persists. Thus, the capacity of a cell to respond and adjust to stress is essential for survival. Because stress responses are critical to cell physiology, the signaling mechanisms that function during stress are highly conserved in eukaryotes. Understanding how stress signaling responses function has considerable human health interest as many diseases result from, or cause cellular stress such as diabetes, heart disease, cancer, and neurodegeneration. In eukaryotes, stresses trigger widespread post-translational modification of proteins with the small ubiquitin-like modifier SUMO. However, the precise functions of protein sumoylation during stress are poorly understood. This deficiency in knowledge is surprising given that SUMO is broadly used as a protein modifier and stress responses are critical for survival. To gain key new insights into SUMO's roles in stress, we initiated new studies in Saccharomyces cerevisiae (budding yeast) to discover specific functions for stress-induced sumoylation. Our studies revealed that hyperosmotic stress triggers the massive, but transient sumoylation of predominantly two proteins: Tup1 and Cyc8. Together, Tup1 and Cyc8 form a transcription corepressor complex that represses the expression of hundreds of genes involved in a wide variety of physiological processes including stress responses. Focusing on Cyc8, we discovered sumoylation of Cyc8 protects against the persistence of stress-induced Cyc8 inclusions in vivo. Because Cyc8 contains a prion domain and is a prionogenic protein, we propose a key function of Cyc8 sumoylation is to maintain Cyc8 solubility and prevent the switch to a persistent aggregation-prone state during hyperosmotic stress. Protection against aggregation is an emerging role for sumoylation, and is especially relevant due to the fact that dysregulation of SUMO signaling occurs in many neurodegenerative diseases associated with protein aggregation. From our recent discoveries, new prominent questions have arisen. 1) How do the signaling mechanisms that respond to hyperosmotic stress control sumoylation of Cyc8 and Tup1? 2) What are the key in vivo and in vitro parameters of Cyc8 sumoylation that inhibit Cyc8 aggregation and inclusion formation? 3) What are the characteristics of Cyc8's prion domain that respond transiently to hyperosmotic stress? These questions form the conceptual basis for the aims in this proposal. We will use a combination of genetics, cell biological assays, mutagenesis experiments, and in vitro biochemistry to address these questions. The proposed studies to dissect the anti-aggregation function of sumoylation during hyperosmotic stress will serve as a paradigm for SUMO`s protective role during stress, especially as it pertains to prionogenic proteins.